ω3 unsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in particular, are known as a functional lipid having physiological effects such as blood lipid reduction and improvement in brain and visual functions. Both EPA and DHA nutrients, which are essential to humans, can be insufficiently provided from foods taken. In order to ensure required dietary intake, health food materials containing EPA and DHA or dietary supplements are widely available. High-purity EPA ethyl ester is used in the form of a medicine such as a hypolipidemic agent. Since health foods containing EPA and DHA ingredients were approved as Foods for Specified Health Use by the Ministry of Health, Labour and Welfare in 2004, the market of ω3 unsaturated fatty acids such as EPA and DHA has been expected to grow, resulting in more widespread commercial use.
Meanwhile, in many cases, it has been reported that a phospholipids containing a fatty acid as a constituent lipid, rather than a fatty acids in itself, includes various useful bioactivities, such as improvement effect of brain function by phosphatidyl serine (PS) (Non-Patent Document 1) and improvement effects of arteriosclerosis and neurological dysfunction by phosphatidyl choline (PC). Recently, not only PS and PC, but also most phospholipids containing phosphatidyl ethanolamine (PE) have received much academic attention to be used in health care dietary supplements.
Under the circumstances, physiological functions such as antitumor and antioxidative properties of a phospholipid comprising an ω3 unsaturated fatty acid as a constituent lipid, for example, PC or PE comprising DHA as a constituent lipid (hereinafter called DHA-PC or DHA-PE, respectively, and most phospholipids containing DHA as a constituent lipid called DHA phospholipids) are being found in not only cases where a cultured cell is used, but also cases where an animal living body is used.
For example, according to a research by Kafrawy, et al. (Non-Patent Document 2), selective cytotoxicity to cancerated animal cells (mouse leukemia cells) is found in DHA-PC, particularly in a molecular species of PC comprising two molecules of DHA (DHAJDHA-PC). Thus, the demand for a phospholipid comprising an ω3 unsaturated fatty acid, particularly DHA, as a constituent lipid, is expected to grow in the future.
DHA phospholipid, which is a major phospholipid comprising an ω3 unsaturated fatty acid as a constituent lipid, is supplied, e.g., mainly from a squid (particularly, skin of neon flying squid), fish oil or eggs from hens produced by providing such fish oil (Patent Document 1). The neon flying squid contains plenty of phospholipids, 50% of which is phosphatidyl choline (PC), whose constituent lipid is DHA (50%), thereby showing a high proportion of DHA phospholipids to the lipid.
However, industrial production of DHA phospholipids, in which marine products such as neon flying squid and fish oil are defined as a source of DHA phospholipids, involves the following problems: unstable DHA phospholipid supply due to variable fish catches, uneven product quality from seasonal and climate changes and unassured product safety due to marine contamination. Additional problems are lower product quality and value due to unpleasant fish odor specific to fish oil and higher costs for refining long-chain highly-unsaturated fatty acids of structural similarity contained in fish oil. In eggs from hens, phospholipid content is high at 30% of yolk lipids, but weight of the total lipids is low. DHA content in ethanolic extract of the yolk is merely about 12%.
A source of an ω3 unsaturated fatty acid other than the above fish oil and eggs from hens is a microorganism capable of producing the ω3 unsaturated fatty acid, particularly a microorganism capable of producing DHA. A method for producing DHA using a microorganism is put into practical use in the United States and such products as ingredients of DHA-containing lipids and high-DHA containing feed are provided into the market. Specifically, a technology for growing genus Thraustochytrium or genus Schizochytrium (Patent Document 2) and a technology for using an ω3 unsaturated fatty acid extracted from Thraustochytriales (Patent Document 3) are developed.
Currently in Japan, various technologies for using labyrinthulean microorganisms as a source of DHA are developed, specifically a technology for using strain S3-2 as a microorganism of genus Labyrinthula (Patent Documents 4 to 6), and strain SR21 as a microorganism of genus Schizochytrium and technology for using it (Patent Documents 7 to 9).
However, all DHAs produced using the above-mentioned microorganisms are a mere DHA as a constituent lipid of fat (triglyceride), neither a constituent lipid of a phospholipid nor a constituent DHA phospholipid.
The inventors isolated a new labyrinthulean microorganism strain 12B as a non-photosynthetic unicellular microorganism to find out its capability of producing a DHA phospholipid and made a patent application (Patent Document 10). Despite this finding, the microorganism can accumulate over 40% DHA of fatty acid of the total lipids, but DHA phospholipid content is merely 12 to 13% of the total lipid of the microorganism.
Most DHA phospholipids prepared from biological materials includes only one molecule of DHA in a phospholipid molecule. In fact, very few biological material-derived phospholipids, in which the content of DHA as a constituent lipid exceeds 50%, are reported. Therefore, improvement in DHA content in a phospholipid is an important issue to increase the value for pharmaceutical use in addition to functional food.